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Abstract:

The present invention provides a self-assembly of a compound or of a
pharmaceutically acceptable salt thereof, the compound being represented
by formula (I)
R1-Gly Leu Ala Met Ala Pro Ser Val Gly His Val Arg Gln His
Gly-R2 (I)
(wherein R1 represents a linear or branched acyl group having 14 to
24 carbon atoms; R2 represents OH, NH2, NR3R4, a
substituted or unsubstituted alkoxy group, a substituted or unsubstituted
aryloxy group, or a substituted or unsubstituted aralkyloxy group; and
R3 and R4 independently represent a substituted or
unsubstituted alkyl group, a substituted or unsubstituted aryl group, a
substituted or unsubstituted aralkyl group, an alkoxy group, or a hydroxy
group, provided that both R3 and R4 do not represent a hydroxy
group or an alkoxy group simultaneously).

Claims:

1. A self-assembly of a compound or of a pharmaceutically acceptable salt
thereof, the compound being represented by the formula I: R1-Gly
Leu Ala Met Ala Pro Ser Val Gly His Val Arg Gln His Gly-R2 (I)
wherein R1 represents a linear or branched acyl group having 16-20
carbon atoms; and R2 represents OH or NH.sub.2.

2. The self-assembly according to claim 1 wherein R1 represents a
stearoyl group, a palmitoyl group, an oleoyl group, or a palmitoleyl.

3. (canceled)

4. The self-assembly according to claim 1 wherein R1 represents a
stearoyl group, and R2 represents OH or NH.sub.2.

5. The self-assembly according to claim 1 wherein the self-assembly is in
the form of a lyophilizate.

6. A prophylactic or therapeutic agent for influenza virus infection
comprising the self-assembly of claim 1.

7. The prophylactic or therapeutic agent according to claim 6 wherein the
influenza virus is of subtype H1, H3, H5, or H7.

8. The prophylactic or therapeutic agent according to claim 6 wherein the
influenza virus is of subtype H1 or H3.

9. The prophylactic or therapeutic agent according to claim 6 wherein the
influenza virus is of subtype H1.

11. A method of preventing or treating influenza virus infection,
comprising administering an effective amount of a compound, a
pharmaceutically acceptable salt thereof, or a self-assembly of the
compound or of the pharmaceutically acceptable salt to a patient infected
with an influenza virus or a subject who is at risk for infection, the
compound being represented by formula R1-Gly Leu Ala Met Ala Pro
Ser Val Gly His Val Arg Gln His Gly-R2 (I) wherein R1
represents a linear or branched acyl group having 16-20 carbon atoms; and
R2 represents OH or NH.sub.2.

Description:

TECHNICAL FIELD

[0001] The present invention relates to a substance having a prophylactic
or therapeutic effect on influenza virus infection, and a prophylactic or
therapeutic agent for influenza virus infection. The present invention
further relates to a method for preventing or treating influenza virus
infection (influenza).

BACKGROUND ART

[0002] Influenza viruses have, in their envelope membranes, two types of
spike glycoproteins, hemagglutinin (HA) and sialidase (NA,
neuraminidase), which play an important role in the establishment of
viral infection and viral budding from a host cell, respectively.

[0003] Hemagglutinin, involved in the first step of influenza virus
infection, has various subtypes, based on the diversity of amino acid
sequences of the antigen-determining regions (A-E), which are highly
mutatable. While amino acid sequence homology among hemagglutinin
subtypes ranges from 25 to 75%, the so-called receptor binding pocket
region, which binds to a receptor of a host cell, is comparatively less
mutatable, and its three-dimensional structure is well-conserved
(Non-patent Literature 1 (NPL 1)).

[0004] To prevent influenza virus infection, research has been undertaken
to develop a vaccine that specifically binds to hemagglutinin that is
contributory to the establishment of an infection, and thereby inhibits
the function of hemagglutinin.

[0005] For example, Patent Literature 1 (PTL 1) discloses a peptide for
preventing influenza virus infection, and particularly discloses a
liposome preparation thereof; and the effects of the liposome preparation
in vitro have been confirmed.

[0006] Patent Literature 2 (PTL 2) demonstrates in FIG. 1 that the peptide
H3G-1 has an inhibitory effect on both H1N1 and H3N2. However, the
inhibitory effect of the peptide H3G-1 was weak.

[0010] An object of the present invention is to provide a substance that
has a high prophylactic or therapeutic effect on influenza virus
infection (influenza).

[0011] Another object of the present invention is to provide a
prophylactic or therapeutic agent for influenza virus.

[0012] Another object of the present invention is to provide a method of
preventing or treating influenza virus.

Solution to Problem

[0013] The present inventors conducted further research on anti-influenza
virus effects of a stearoylated peptide as set forth in SEQ ID NO: 1. As
a result, the inventors found that the peptide in the form of a liposome
preparation exerts an anti-influenza virus effect only on H3N2, whereas a
self-assembly of the peptide has greater effectiveness for a wide range
of influenza viruses, such as subtypes H1, H3, H5, and H7. The present
inventors further found that the self-assembly of the peptide can be
obtained by acylating the N-terminus of the peptide with an acyl group
having 14 to 24 carbon atoms.

[0014] The present invention provides the following self-assembly and
prophylactic or therapeutic agent for influenza virus infection.

Item 1. A self-assembly of a compound or of a pharmaceutically acceptable
salt thereof, the compound being represented by the formula I:

R1-Gly Leu Ala Met Ala Pro Ser Val Gly His Val Arg Gln His
Gly-R2 (I)

(wherein R1 represents a linear or branched acyl group having 14 to
24 carbon atoms; R2 represents OH, NH2, NR3R4, a
substituted or unsubstituted alkoxy group, a substituted or unsubstituted
aryloxy group, or a substituted or unsubstituted aralkyloxy group; and
R3 and R4 independently represent a substituted or
unsubstituted alkyl group, a substituted or unsubstituted aryl group, a
substituted or unsubstituted aralkyl group, an alkoxy group, or a hydroxy
group; provided that both of R3 and R4 do not represent a
hydroxy group or an alkoxy group simultaneously). Item 2. The
self-assembly according to Item 1 wherein R1 represents a stearoyl
group, a palmitoyl group, an oleoyl group, or a palmitoleyl. Item 3. The
self-assembly according to Item 1 wherein R2 represents OH or
NH2. Item 4. The self-assembly according to Item 1 wherein R1
represents a stearoyl group, and R2 represents OH or NH2. Item
5. The self-assembly according to any one of Items 1 to 4 wherein the
self-assembly is in the form of a lyophilizate. Item 6. A prophylactic or
therapeutic agent for influenza virus infection comprising the
self-assembly of any one of Items 1 to 5. Item 7. The prophylactic or
therapeutic agent according to Item 6 wherein the influenza virus is of
subtype H1, H3, H5, or H7. Item 8. The prophylactic or therapeutic agent
according to Item 6 wherein the influenza virus is of subtype H1 or H3.
Item 9. The prophylactic or therapeutic agent according to Item 6 wherein
the influenza virus is of subtype H1. Item 10. A compound, a
pharmaceutically acceptable salt thereof, or a self-assembly of the
compound or of the pharmaceutically acceptable salt, the compound being
represented by formula I:

R1-Gly Leu Ala Met Ala Pro Ser Val Gly His Val Arg Gln His
Gly-R2 (I)

(wherein R1 represents a stearoyl group, and R2 represents
NH2). Item 11. A method of preventing or treating influenza virus
infection, comprising administering an effective amount of a compound, a
pharmaceutically acceptable salt thereof, or a self-assembly of the
compound or of the pharmaceutically acceptable salt to a patient infected
with an influenza virus or a subject who is at risk for infection, the
compound being represented by formula I:

R1-Gly Leu Ala Met Ala Pro Ser Val Gly His Val Arg Gln His
Gly-R2 (I)

(wherein R1 represents a linear or branched acyl group having 14 to
24 carbon atoms; R2 represents OH, NH2, NR3R4, a
substituted or unsubstituted alkoxy group, a substituted or unsubstituted
aryloxy group, or a substituted or unsubstituted aralkyloxy group; and
R3 and R4 independently represent a substituted or
unsubstituted alkyl group, a substituted or unsubstituted aryl group, a
substituted or unsubstituted aralkyl group, an alkoxy group, or a hydroxy
group; provided that both of R3 and R4 do not represent a
hydroxy group or an alkoxy group simultaneously).

Advantageous Effects of Invention

[0015] According to the present invention, a potent prophylactic or
therapeutic agent for influenza/influenza virus infection can be
obtained.

[0016] The peptide set forth in SEQ ID NO: 2 exhibits a potent effect in
vitro. However, this peptide is ineffective in vivo, and actually
exacerbates influenza virus infection in vivo.

[0017] In contrast, the N-acylated peptides represented by formula (I) of
the present invention, particularly the peptide set forth in SEQ ID NO:
2, exhibit a potent prophylactic or therapeutic effect on influenza
viruses in vivo as well as in vitro.

[0018] Furthermore, C18D1 (stearoyl-GLAMAPSVGHVRQHG-NH2), when formed
into a liposome, has a very low IC50 value of about 500 nM against
influenza viruses H1 and H3, and exhibits substantially no anti-influenza
virus effect; whereas a self-assembly of C18D1 exhibits a highly potent
effect.

BRIEF DESCRIPTION OF DRAWINGS

[0019] FIG. 1 shows the survival benefit achieved by administering the
peptide C18-D1 to influenza virus-infected mice. C18-D1
(C18-GLAMAPSVGHVRQHG-NH2) and C18-S2 (C18-ARLPRTMVHPKPAQP-NH2)
were used. The dose of the peptide C18-D1 was 17.4 mg/(kg), and the dose
of the peptide C18-S2 was 19.4 mg/(kg).

DESCRIPTION OF EMBODIMENTS

[0020] According to the present invention, a self-assembly of the compound
represented by formula I shown below or a pharmaceutically acceptable
salt thereof can be used as a prophylactic or therapeutic agent for
influenza virus infection.

R1-Gly Leu Ala Met Ala Pro Ser Val Gly His Val Arg Gln His
Gly-R2 (I)

(wherein R1 represents a linear or branched acyl group having 14 to
24 carbon atoms; R2 represents OH, NH2, NR3R4, a
substituted or unsubstituted alkoxy group, a substituted or unsubstituted
aryloxy group, or a substituted or unsubstituted aralkyloxy group; and
R3 and R4 independently represent a substituted or
unsubstituted alkyl group, a substituted or unsubstituted aryl group, a
substituted or unsubstituted aralkyl group, an alkoxy group, or a hydroxy
group; provided that both of R3 and R4 do not represent a
hydroxy group or an alkoxy group simultaneously).

[0021] When the compound represented by formula I according to the present
invention is formed into a self-assembly, the self-assembly exhibits an
increased prophylactic or therapeutic effect on influenza virus
infection. Furthermore, the self-assembly can gain effectiveness for a
wide range of influenza viruses, such as subtype H1 (for example, H1N1,
H1N2), subtype H3 (for example, H3N2), subtype H5 (for example, H5N1),
and subtype H7 (for example, H7N1).

[0022] The effectiveness of the self-assembly of the present invention for
subtypes H1 and H3 is demonstrated, as shown in the Examples below. Since
the self-assembly of the compound represented by formula I is effective
against both subtypes H1 and H3, the self-assembly is assumed to bind to
a receptor-binding pocket region of a host cell, which is comparatively
less mutatable and whose three-dimensional structure is well-conserved,
thereby exhibiting an anti-influenza virus effect. Accordingly, the
self-assembly of the compound represented by formula I according to the
present invention is effective against infection of subtypes H1 and H3 as
well as subtypes H5 and H7, which all have a common receptor-binding
pocket region.

[0023] The self-assembly of the present invention is effective in vivo as
well as in vitro. For example, C18-S2 (stearoyl
ARLPRTMVHPKPAQP-NH2), obtained by stearoylation of the N-terminus of
S2 (ARLPRTMVHPKPAQP-NH2), exhibits a potent anti-influenza virus
effect in vitro. However, C18-S2 is ineffective in vivo, and actually
causes early mortality due to influenza virus infection. Thus, the
anti-influenza virus effect of C18-S2 is greatly different in vivo and in
vitro. One feature of the peptide according to the present invention is
that the peptide of the invention exhibits an anti-influenza virus effect
in vivo as well as in vitro.

[0024] The self-assembly according to the present invention has a
micelle-like structure wherein an acyl group moiety represented by
R1 is directed inward, and a peptide moiety is directed outward.
Since a liposome preparation is in the form of a liposome containing a
phospholipid as a major component, a self-assembly of the compound
represented by formula I cannot be formed. Although Patent Literature 1
discloses an N-terminus-stearoylated (C18) peptide having a carboxyl
group (COOH) on the C-terminus in the Examples, a stearoylated peptide
having an amide (CONH2) on the C-terminus according to the present
invention is not disclosed therein.

[0025] Patent Literature 1 only discloses the production of a compound
represented by formula I wherein R1 represents a stearoyl group and
R2 represents OH, and a liposome preparation containing the
compound. Patent Literature 1 does not disclose a self-assembly. While
not wishing to be bound by theory, the present inventors believe that the
effectiveness of the self-assembly of the compound of formula I according
to the present invention for a wide range of influenza viruses is
attributable to the structure of the self-assembly, because Patent
Literature 1 demonstrates that the liposome preparation containing the
compound of formula I is effective only for subtype H3, and is
ineffective for subtype H1 (H1N1).

[0026] The N-terminus-non-acylated peptide of formula (I) (wherein R1
is H), shown as H3G-1 in FIG. 1 of Patent Literature 2, exhibits an
IC50 value of >100 μM in a plaque assay of the influenza virus
H1N1, and thus has a very weak influenza virus infection-inhibitory
effect. The inventors believe that the self-assembly structure is
important because the peptide of formula (I) wherein R1 is H cannot
form a self-assembly.

[0027] The self-assembly of the compound of formula I can be produced by
dissolving the compound of formula I in water, optionally followed by
stirring. With respect to the concentration in an aqueous solution to
form a self-assembly, because the critical micelle concentration is about
0.5 to about 2 μM (for example, about 1.3 μM, when R1 is a
stearoyl group), a self-assembly can be produced by dissolving the
compound of formula I in a concentration not lower than the critical
micelle concentration. The higher the concentration of the compound of
formula I, the larger the average particle diameter of the self-assembly
tends to be. The average particle diameter of the self-assembly of the
compound of formula I is preferably about 100 nm or more; and may be, for
example, about 100 nm to about 20 μm, preferably about 300 nm to about
10 μm, more preferably about 500 nm to about 7 μm, and particularly
preferably about 700 nm to about 5 μm.

[0028] The self-assembly as used herein may be obtained by dissolving the
compound in water, i.e., in the form of a dispersion in water. Examples
of the self-assembly include a wide range of products that can easily
reproduce a self-assembly when brought into contact with water, such as
lyophilizates thereof.

[0029] Examples of the acyl group as used herein includes O14-24
linear or branched acyl groups that may be substituted with a hydroxy
group, such as a myristoyl group, a palmitoyl group, a stearoyl group, an
arachidoyl group, a behenoyl group, a lignoceroyl group, a myristoleoyl
group, a palmitoleoyl group, an oleoyl group, a linoleoyl group, a
γ-linolenoyl group, an α-linolenoyl group, an arachidoyl
group, an elaidoyl group, an eicosatrienoyl group, an isostearoyl group,
a 12-hydroxystearoyl group, a ricinoleoyl group, and the like.

[0031] Because the compound of formula I wherein R1 is H is not
capable of forming a self-assembly, the compound exhibits a very weak
anti-influenza virus effect, compared to the compound of the present
invention. Because the compound of formula I wherein R1 is a lauryl
group (C12) has a low self-assembly-forming capability, the compound
exhibits very weak activity in in vitro tests. In contrast, a polypeptide
wherein R1 contains 14 carbon atoms (myristoyl) has an intermediate
level of activity. A polypeptide wherein R1 contains 16 to 22 carbon
atoms has potent activity. When R1 contains more than 20 carbon
atoms, the obtained peptide has low water solubility. Therefore, R1
preferably contains 16 to 20 carbon atoms, and particularly 18 to 20
carbon atoms.

[0032] Examples of the alkyl group as used herein include alkyl groups
containing 1 to 6 carbon atoms, and preferably 1 to 4 carbon atoms, such
as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,
tert-butyl, pentyl, and hexyl. Examples of substituents of the alkyl
group include a hydroxy group, a fluorine atom, a methoxy group, an
ethoxy group, and the like.

[0035] Examples of the alkoxy group include O-(alkyl). The alkyl group is
as defined above. Examples of the substituent of the alkoxy group include
a hydroxy group, a fluorine atom, a methoxy group, an ethoxy group, and
the like.

[0036] Examples of the aryloxy group include O-(aryl). The aryl group is
as defined above.

[0037] Examples of the aralkyloxy group include O-(aralkyl). The aralkyl
group is as defined above.

[0038] Examples of substituents of the alkyl group include a fluorine
atom, an alkoxy group, a cyano group, a hydroxy group, and the like.

[0039] Examples of substituents of the aryl group, aralkyl group, aryloxy
group, or like aryl moiety; and substituents of the aralkyloxy group or
like aryl moiety include halogen atoms (F, Cl, Br, I), nitro, amino,
monoalkylamino, dialkylamino, acetyl, methylenedioxy, alkyl, alkoxy,
carbamoyl, acetylamino, and the like. The number of substituents is 1 to
5, preferably 1 to 3, and more preferably 1 to 2.

[0041] The compound of formula I as used herein can be obtained by:
synthesizing a compound represented by formula I wherein R1 is a
hydrogen atom, and R2 is a group as defined above (H-Gly Leu Ala Met
Ala Pro Ser Val Gly His Val Arg Gln His Gly-R2) by liquid phase
synthesis or solid phase synthesis according to a usual peptide synthesis
method, such as the Fmoc method; and introducing an acyl group to R1
by using a coupling reagent such as DIC. The R2 group can be
introduced by using Gly-R2 as an amino acid on the C-terminus. The
R2 group can be excised as an OH or NH2 peptide by suitably
selecting a carrier for solid phase synthesis.

[0043] The self-assembly of the present invention can be formulated into a
pharmaceutical preparation comprising the self-assembly of the present
invention as an active ingredient, and optionally other components such
as pharmacologically acceptable carriers, diluents, and excipients. The
pharmaceutical preparation can be administered to, for examples, infected
or uninfected humans or other mammals, birds, etc.

[0044] The method of administering the prophylactic or therapeutic agent
for influenza virus infection according to the present invention is not
particularly limited, and can be suitably selected according to the form
of the pharmaceutical preparation; the age, sex, and other conditions of
the patient; the severity of the disease, etc. Examples of preferable
dosage forms are oral preparations such as tablets, capsules, granules,
and sublingual tablets; and parenteral dosage forms such as injections,
drips, nasal drops, inhalants, patches, and poultices. Inhalants are
particularly preferable.

[0045] The daily dose of the self-assembly of the compound of formula I
used as an active ingredient of the prophylactic or therapeutic agent for
influenza according to the present invention (including pharmaceutical
compositions and pharmaceutical preparations) may vary depending on the
subject's condition, body weight, age, sex, etc., and cannot be
completely generalized. However, the dose can typically be selected from
the range of about 0.001 to about 100 mg per day per adult. The
prophylactic or therapeutic agent for influenza can be administered once
a day, or in divided doses.

[0046] The present invention is described below in further detail with
reference to Reference Examples and Examples, but is not limited thereto.

EXAMPLES

Reference Example 1

[0047] A polypeptide represented by H-Gly Leu Ala Met Ala Pro Ser Val Gly
His Val Arg Gln His Gly-NH2 was synthesized by solid phase synthesis
using Fmoc amino acid (1.4 equivalents), HOBt (1-hydroxybenzotriazole;
2.5 equivalents), and DIC (2.8 equivalents) according to the Fmoc method.
The obtained polypeptide was allowed to react in a mixed solvent of DMF
and DCM using stearic acid (1 equivalent), DIC (2.8 equivalents), and
HOBt (2.5 equivalents) under conditions similar to the peptide synthesis
for 90 minutes to synthesize a peptide of the present invention
represented by (stearoyl)-Gly Leu Ala Met Ala Pro Ser Val Gly His Val Arg
Gln His Gly-NH2 that has a strearoyl group bound to the amino group
at the N-terminus (hereinafter referred to as "C18-D1"). C18-D1 was
purified by HPLC using a C-18 column. The production of C18-D1 was
confirmed by mass spectrometry ([M+H].sup.+=1782.29).

Reference Example 2

[0048] C18-S2 (stearoyl ARLPRTMVHPKPAQP-NH2), S2
(ARLPRTMVHPKPAQP-NH2), and D1 (GLAMAPSVGHVRQHG-NH2) were
synthesized in a manner similar to Reference Example 1. The production of
C18-S2, S2, and D1 was confirmed by mass spectrometry.

[0050] The critical micelle concentration (CMC) of the purified C18-D1 was
determined in the following manner. PBS (pH 7.5) containing 1 μM of
N-phenyl-1-naphthylamine (hereinafter abbreviated as "NPN") was prepared.
A C18-D1 peptide stock solution (1 mM) was serially diluted to final
concentrations of 0.1, 0.3, 1.3, 10, and 30 μM. These solutions were
excited by a wavelength of 350 nm, and the fluorescence intensity (FI) at
450 nm was measured. The difference in FI between each diluted peptide
solution and PBS alone was plotted as the ordinate, and the peptide
solutions were plotted as the abscissa. The concentration at the
intersection of the high concentration curve and the low concentration
curve was determined. The critical micelle concentration (CMC) of the
intersection was 1.3 μM.

Example 2

In Vitro Test for the Inhibition of Influenza Infection

[0051] Inhibition of influenza virus infection was determined by a plaque
assay on MDCK cells. The MDCK cells in 6-well plates were incubated with
0.2 mL of influenza virus A/PR/8/34 solutions (100 to 200 pfu; pfu
denotes plaque-forming units; subtype H1N1) or influenza virus
A/Victria/1/75 solutions (100-200 pfu; subtype H3N2) each containing
C18-D1, S2 (H-ARLPRTMVHPKPAQP-NH2), C18-S2
(stearoyl-ARLPRTMVHPKPAQP-NH2), or D1 (H-GLAMAPSVGHVRQHG-NH2).
After incubation at 37° C. under 5% CO2 for 30 minutes, the
supernatant was removed, and the cells were washed with PBS. 2 mL (per
well) of 2×MEM+BSA containing 0.6% agarose, 0.01% of
O-diethylaminoethyl cellulose dextran, 0.1% NaHCO3, and 0.01
μg/mL of acetyl trypsin was added, and the cells were incubated for
two days. Viable cells were stained with a crystal violet solution (1
mg/mL in 20% ethanol), and the number of plaques was counted. The maximum
infection activity (100%) was defined as the number of plaques in the
case of the absence of C18-D1. The IC50 value (50% inhibitory
concentration) of C18-D1 was obtained from the plot between log [f/(1-f)]
and log [C18-D1], wherein f is the percent infection activity. The
IC50 values of S2, C18-S2, and D1 were calculated in a similar
manner.

[0052] The results of the investigation of influenza virus infection
inhibitory effects of compound A on MDCK cells are as follows.

[0053] Influenza virus infection inhibitory effect in vivo of C18-D1 on
MDCK cells was determined by the plaque assay. Infection of MDCK cells
with influenza type A virus HAl-- (A/PR/8/34 (H1N1)) and influenza
type A virus HA3 (A/Victria/1/75 (H3N2)) was inhibited in the presence of
C18-D1.

[0054] As shown in Table 1, the self-assembly of 018-D1 of the present
invention and C18-S2 exhibited excellent anti-influenza virus activity in
vitro.

Example 3

Animal Test for Inhibition of Influenza Infection

[0055] An experiment was conducted to investigate the virus infection
inhibitory effect of C18-D1 of the present invention and C18-S2 on mice.
Peptide stock solutions (2.5-7.5 mM in PBS), and PBS containing influenza
virus (H1N1) (200 pfu) were mixed in the amounts described below, and
allowed to stand at room temperature for 30 minutes. The samples thus
prepared were administered intranasally to mice (50 μL per mouse).

[0056] More specifically, solutions each containing 200 pfu/50 μL of
influenza virus, and C18-D1 or C18-S2 in a final concentration of 3.75 mM
or a solvent (PBS, mPR8) were administered. When C18-D1 was administered,
80% or more of mice survived (FIG. 1), thus indicating that C18-D1 has
infection inhibitory activity. In contrast, administration of C18-S2,
which exhibited activity equal to or better than C18-D1 in the in vitro
test, caused early mortality in the in vivo test, even compared to the
control.

[0057] The results thus show that in vivo data of self-assemblies cannot
be estimated from in vitro data thereof, thus demonstrating an
unpredictable effect of the self-assembly of the present invention.

[0058] When only the virus (mPR8) was administered, all of the mice died.

[0059] The term "mPR8" indicates a control prepared by mixing the
influenza virus with PBS.